A rapid real-time quantitative PCR method was developed for
V. parahaemolyticus in sediment of the Stichopus japonicus culture pond by using specific primers targeted toxR gene. The known number V. parahaemolyticus cells were spiked into sterile sediment served as the simulative sediment samples. Three modified sediment DNA extraction methods were tried, it was proved that modified lysozyme-SDS gentle lyse method is feasible for the sediment DNA extraction. The standard curves was made by using serial diluted total DNA extracted from the simulated sediment and which from plasmid of pure culture. Besides, sensitivity, specificity and repeatability of the detection method were demonstrated well. The quantification limit was found to be 102 cfu g-1 for V. parahaemolyticus in sediment, and have a high repeatability as a result of all the coefficient of variation (CV) values between 0.31% and 0.92%, less than 5%. Development of the real-time PCR quantification method for V. parahaemolyticus in the sediment was important for disease prevention the cultured Stichopus japonicas.
toxR, Vibrio parahaemolyticus, Real-time Quantitative PCR, Stichopus japonicus culture pond, Sediment DNA extraction method
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